Ionic mechanisms of regulatory volume increase (RVI) in the human hepatoma cell-line HepG2

Abstract.

We studied the effects of hypertonic stress on ion transport and cell volume regulation (regulatory volume increase; RVI) in the human tumor cell-line HepG2. Ion conductances were monitored in intracellular current-clamp measurements with rapid ion-substitutions and in whole-cell patch-clamp recordings; intracellular pH buffering capacity and activation of Na⁺/H⁺ antiport were determined fluorometrically; the rates of Na⁺-K⁺-2Cl^– symport and Na⁺/K⁺-ATPase were quantified on the basis of time-dependent and furosemide- or ouabain-sensitive ⁸⁶Rb⁺ uptake, respectively; changes in cell volume were recorded by means of confocal laser-scanning microscopy. It was found that hypertonic conditions led to the activation of a cation conductance that was inhibited by Gd³⁺, flufenamate as well as amiloride, but not by benzamil or ethyl-isopropyl-amiloride (EIPA). Most likely, this cation conductance was non-selective for Na⁺ over K⁺. Hypertonic stress did not change K⁺ conductance, whereas possible changes in Cl^– conductance remain ambiguous. The contribution of Na⁺/H⁺ antiport to the RVI process appeared to be minor. Under hypertonic conditions an approximately 3.5-fold stimulation of Na⁺-K⁺-2Cl^– symport was observed but this transporter did not significantly contribute to the overall RVI process. Hypertonic stress did not increase the activity of Na⁺/K⁺-ATPase, which even under isotonic conditions appeared to be working at its limit. It is concluded that the main mechanism in the RVI of HepG2 cells is the activation of a novel non-selective cation conductance. In contrast, there is little if any contribution of K⁺ conductance, Na⁺/H⁺ antiport, Na⁺-K⁺-2Cl^– symport, and Na⁺/K⁺-ATPase to this process.